PROJECT SUMMARY Diffuse gliomas, including oligodendroglioma, astrocytoma, and the highly malignant glioblastoma (GBM), are the most common type of primary malignant brain tumor. Increased activation of the phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR signaling pathway is common in GBM and appears to be one mechanism of malignant transformation of tumors from lower grade diffuse glioma to GBM. Given the importance of this pathway in disease, several therapeutic strategies that target it are being tested in early phase clinical trials. To maximize benefit and minimize toxicity to these therapies, however, accurate and robust biomarkers for patient stratification and assessment of disease response to therapy are needed. Analysis of protein phosphorylation provides integrated, functional information about signaling pathway activity. The goal of this proposal is to use biospecimen sciences to identify and mitigate the preanalytic factors, including intratumoral heterogeneity, tissue acquisition, and confounding non-neoplastic cells, that can obscure accurate analysis of protein phosphorylation in diffuse glioma. We hypothesize that accurate assessment will require small, well-preserved and biologically targeted tumor biopsies. To investigate this hypothesis we propose a multidisciplinary team of investigators with expertise in the acquisition and analysis of multiple and image-guided biopsies, brain tumor biology, biobanking, neuropathology, imaging, biostatistics, and neuro-oncology. First, we investigate innovative approaches to target tumor biopsies to biologically aggressive tumor regions using pre-operative MR spectroscopic imaging and intra-operative tumor metabolism using 5-aminolevulinic acid (ALA)-based imaging. Second, we use clinically validated assays and state-of-the-art single cell mass cytometry to evaluate PI3K/AKT/mTOR signaling pathway activity and determine the contribution of non-neoplastic cells to overall phosphoprotein levels. Third, using the evidence-based methodologies and approaches we develop, we will investigate the prognostic significance of PI3K/AKT/mTOR signaling pathway activity as determined by protein phosphorylation in diffuse glioma and in patients treated with an mTOR inhibitor by integration with our Phase 2 clinical trial targeting the PI3K/AKT/mTOR signaling pathway (NCT02023905).